An ideal tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, would be a highly effective vaccine that prevents blood stage infection and thereby prevents both disease and transmission. Sanaria's goal is to develop and commercialize a Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in > 90% of recipients. The Sanaria(R) PfSPZ Vaccine is composed of attenuated, purified, aseptic, cryopreserved PfSPZ. The platform technology developed to manufacture the vaccine has also allowed the manufacture of PfSPZ for challenge infections to test vaccines and drugs (PfSPZ Challenge) and for vaccination by challenge under chloroquine protection (PfSPZ-CVac). These comprise a portfolio of products that are on an aggressive timeline to commercialization. The PfSPZ used in these products are extracted from aseptically reared mosquitoes. Increasing the number of SPZ per mosquito directly reduces the cost of goods. This project will develop a strain of genetically modified (GM) Anopheles stephensi mosquitoes highly susceptible to PfSPZ infections. By silencing a mosquito immune effector gene, LRIM1 (a member of the LRR gene family), we have reproducibly increased SPZ loads in A. stephensi. We have also demonstrated a phenotype of increased oocyst burdens in a transgenic A. stephensi strain expressing a SRPN6 double- stranded RNA (dsRNA) sequence in a hairpin construct that was fully integrated into the genome. These major results justify this Phase II project which will create GM A. stephensi lines in which expression of LRIM1 or APL1 is reduced or eliminated, providing a stable and efficient SPZ-production platform for Sanaria's manufacturing process. Two strategies are planned for silencing LRIM1: 1) directly silencing the genes through the expression of gene-specific dsRNA to elicit an endogenous RNA interference response against LRIM1 or APL1 transcripts; 2) indirectly silencing the genes by disrupting the relevant regulatory signaling pathway through either the over-expression of the negative-regulatory protein Caspar or a dominant-negative variant of the transcription factor Rel2. Our successful Phase I strategy was to directly silence genes by driving the expression of gene-specific dsRNA to elicit endogenous RNA interference; this approach will be improved upon for the creation of GM A. stephensi. We will create gene-silencing transgenes by incorporating the GAL4/UAS binary expression system and a 'two-target' gene silencing technology. This strategy will enable us to limit the costly and time consuming assessments of infection phenotype as it enables efficient determination of gene silencing prior to infectious feeding. This approach will enhance our capacity to produce strains with the optimal genotypes and phenotypes. We will screen GM lines to identify those that consistently produce e 2 fold more PfSPZ compared to the current production strain of A. stephensi. Selected strains will be studied under standard insectary conditions, and under the aseptic conditions used for GMP compliant manufacturing. We will incorporate the optimal GM strain of A. stephensi into Sanaria's manufacturing process. PUBLIC HEALTH RELEVANCE: To protect individuals against malaria and to aid in eradicating this disease, a highly effective vaccine is needed. Sanaria has developed a platform technology, manufacture of live Plasmodium falciparum sporozoites (PfSPZ) to make such a vaccine. Two are in clinical development. The first is a live attenuated whole organism vaccine called the PfSPZ Vaccine. The second is a live infectious vaccine administered with an antimalarial drug, a vaccine called PfSPZ-CVac. In addition Sanaria has developed live, infectious PfSPZ, a product called PfSPZ Challenge, for use in testing malaria drugs and vaccines. All the products require the growth of PfSPZ in aseptic Anopheles stephensi mosquitoes. This proposal aims to increase the yields of sporozoites by genetically modifying the mosquito immune system. Success in this project will greatly reduce cost of vaccine manufacture, thereby making the vaccine available more cheaply for travelers and developing world populations.